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A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells
JoVE Journal
Genética
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JoVE Journal Genética
A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

DOI:
10.3791/57413-v

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11:58 min

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March 05, 2018

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,
1Department of Pharmacology,University of Minnesota

Capítulos

  • 00:04Título
  • 01:18Generation of DNA-protein Crosslink Containing Plasmid
  • 06:21Transfection into Mammalian Cells
  • 08:39SSPE-qPCR
  • 10:02Results: Use of Strand-specific Primer Extension Based q-PCR to Quantify the DNA-protein Crosslinks
  • 11:08Conclusion

Summary

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The goal of this protocol is to quantify the repair of defined DNA-protein crosslinks on plasmid DNA. Lesioned plasmids are transfected into recipient mammalian cell lines and low-molecular weight harvested at multiple time points post-transfection. DNA repair kinetics are quantified using strand-specific primer extension followed by qPCR.

Tags

DNA-protein CrosslinksPlasmid DNAMammalian CellsDNA RepairTrans-Pacific Primer Extension QPCR AssayAdductsPolymerase BlockingHouse Cell Reactivation8-oxoguanineT4 Polynucleotide KinasePrimer ExtensionPhenol-chloroform Extraction

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